DESCRIPTION: The focus of this study is the defense response of the model plant Arabidopsis thaliani to the phytopathogenic bacteria Pseudomonas syringae. There are two major goals of this competing renewal application. First, the investigator proposes to further define the incompatible plant resistance response to an avirulent P. syringae strain through the identification of plant genes that act downstream of the RPS2 resistance gene. A second major goal is to dissect the compatible interactions between Arabidopsis and the virulent pathogen P. syringae through the detailed molecular analysis of many mutants that are altered in this response. Avirulent strains of A. syringae, unlike virulent strains, possess an avirulence gene (avrRpt2) whose product is either directly or indirectly recognized by the Arabidopsis resistance gene RPS2. This so-called gene-for-gene interaction leads to the hypersensitive response (HR) in the plant which includes the induction of many genes, the synthesis of plant antibiotics (phytoalexins), oxidative bursts, the induction of systemic resistance (SAR) and cell death at the site of pathogen entry. The cloning of the first resistance (R) genes in this and in other labs led to the discovery of a related group of plant proteins that can recognize bacterial, fungal and virally encoded avirulence genes and trigger the HR. The characterization of the interaction between RPS2 and avrRpt2 is not a focus of this proposal; that project will be undertaken by the postdoctoral who isolated RPS2 and now has his own lab. Rather, one of the Specific Aims involves the identification of putative genes in the signal transduction pathway downstream of RPS2. Initial screens of mutagenized resistant plants in two labs identified eight susceptible mutants - all allelic to RPS2. By refining this screen through the use of mutagenized resistant plants that now carry a second copy of RPS2 as a transgene, it is hoped that additional genes required for resistance will be identified. A second screen for similar genes involves the identification of Arabidopsis mutants that aberrantly express one of two reporter constructs (AIG1-GUS or GST1-GUS) which are both induced by the avrRpt1-RPS2 interaction. Although both cis- and trans-mutations will be isolated, the desired trans-mutations should be easily discerned by assaying the behavior of the endogenous AIG1 and GST1 genes. The promoters of these genes are differentially induced and, thus, may select for different trans-acting regulators. Demonstration that GST1 is downstream of the oxidative burst associated with the HR may provide the first genetic handle on this aspect of the defense response. The second aim involves the characterization of previously isolated Arabidopsis mutants that are altered in their response to the virulent strain Psm ES4326. In a screen for enhanced susceptiblity to this virulent pathogen, 26 mutants were isolated that fall into two classes, those that permit more bacterial growth (eds) and those that do not (ens). The existence of ens mutants suggest that symptom development during a compatible interaction does not simply reflect bacterial growth but rather the ability of the plant to recognize the bacteria and trigger a response such as programmed cell death. Complementation analysis reveals the existence of numerous loci and indicates that the screen is not saturated. Some mutants are allelic to previously characterized pad (phytoalexin deficient) and npr (non-expressors of pathogen resistance genes) mutants. Mutants will be divided between the investigator and the lab of Dr. Glazebrook, a former postdoctoral now at the U. of Maryland where they will be characterized and mapped and a representative from each group crossed into Landsberg erecta for mapping. Previously isolated mutants that display reduced symptoms after infiltration of PsmES4326 either due to reduced bacterial growth (reduced disease susceptibility, rds) or not (reduced symptoms, res) will be tested to see if they have a cpr phenotype (constitutive induction of PR genes) by assaying an inducible reporter gene already in the strain. cpr mutants will be characterized in collaboration with a former postdoc, Dr. Dong, now at Duke. Mutant phenotypes will also be tested for their dominant or recessive behavior and segregation as single Mendelian loci as will the possibility that res mutants are weak alleles of rds mutants. The defense-related genes characterized in Aims 1 and 2 will be assigned to signal transduction pathways by (I) testing their general susceptiblity to other known bacterial and fungal pathogens of Arabidopsis, (II) monitoring their defects in various responses, (III) testing their response to mutant bacteria altered in pathogenic traits and (IV) constructing double mutants. The function of mutant genes will be addressed by determining whether any contain mutations in known pathogen-induced genes. A complementary approach will involve the assignment of phenotypes to known pathogen-induce genes via the identification of insertions into these genes or their knock out by antisense stragegies. Finally, selected defense-related genes will be cloned using map-based cloning strategies.